Increased airway smooth muscle mass is thought to contribute to the airway hyperresponsiveness observed in patients with asthma. The potential importance of abnormal airway smooth muscle cell proliferation highlights the need for an understanding of the early events involved in airway smooth muscle mitogenesis. To achieve this overall goal, we propose the following Specific Aims:
SPECIFIC AIM 1 : Determine the precise role of cyclin D(1) in bovine tracheal myocyte G(1) traversal. We will assess cyclin D(1) expression and cyclin D(1)-dependent kinase activity following mitogenic stimulation by immunoblotting and measurement of retinoblastoma protein phosphorylation. The requirement and sufficiency of cyclin D(1) for bovine tracheal myocyte cell cycle progression will be assessed by stable transfection of cells with either cyclin D(1) antisense or sense cDNA using the inducible TET repressor system, followed by flow cytometry and cell counting. The effects of TGFbeta and forskolin, an activator of adenylate cyclase, on cyclin D(1) synthesis, associated kinase activity and cdk inhibitor expression will be determined.
SPECIFIC AIM 2 : Identify downstream targets of MAPK in bovine tracheal myocytes. We will assess cyclin D(1) promoter transcriptional activity in cells transiently- transfected with the cyclin D(1) promoter, subcloned into a luciferase reporter. Luciferase activity will be measured after stimulation with mitogens and non-mitogens, as well as following co-transfection with plasmids encoding dominant-negative or constitutively-active forms of MEK- 1, a dual-function kinase required and sufficient for MAPK activation in these cells. The cis-acting DNA sequences required for MAPK-induced cyclin D(1) promoter activity will be assessed with a series of cyclin D(1) 5' flanking region deletion mutants. We will identify the precise nuclear transcription factor(s) involved in this transcriptional regulation by performing gel mobility shift assays, DNase footprinting and Southwestern blotting.
SPECIFIC AIM 3 : Identify alternative, Raf-1- independent upstream activation pathways of MAPK in bovine tracheal myocytes. Kinase activity for MEK-1 will be isolated from forskolin and PDGF-treated cells using a Mono-Q column and assessed by in vitro phosphorylation assay. Fractions with kinase activity will be probed with antibodies against MEK kinase (MEKK), Raf-1, A-Raf, B-Raf and Mos. If detected by immunoblotting, the role of the MEK activator(s) will be confirmed by sequential immunoprecipitation, followed by re-testing for MEK kinase activity. If the MEK activator is novel, it will be purified by column chromatography, visualized by active-site labeling, microsequenced and cloned. This work may shed light on parallel mechanisms that may operate in asthma, and lead to therapeutic interventions.
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